Project Summary/Abstract Proteolysis by the ubiquitin system plays major roles in a multitude of biological processes. Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. This is renewal application for the DK39520 grant, currently in its 24th year of support. Two of several discoveries during the preceding period of support are the new functions of the N-end rule pathway as a sensor, through different mechanisms, of both heme and nitric oxide. Another discovery is the identification and analysis of an entirely new class of aminoacyl-transferases that underlie the N-end rule pathway in prokaryotes. A focus of the present renewal application is on physiological substrates, mechanisms and functions of ubiquitin ligases in both yeast and mammalian N-end rule pathways. Hence the (overdue) change of DK39520 title, from [unreadable]Mechanics and Functions of the Yeast N-End Rule Pathway[unreadable] to [unreadable]Ubiquitin Ligases, Mechanisms and Functions of the N-End Rule Pathway[unreadable]. This new title more accurately describes the scope (including the range of model organisms) of our research supported by the DK39520 grant. Specific Aims: 1) Studies of S. cerevisiae MGT1, a key DNA repair enzyme that we recently identified as a physiological substrate of the N-end rule[unreadable]s Ub ligase UBR1. Extension of these insights to MGMT, a mammalian DNA repair enzyme (the counterpart of MGT1), and to N-end rule[unreadable]s Ub ligases of mammals. 2) Systematic applications of our new in vivo method for discovering N-end rule substrates. This method is based on the use of altered genetic code, a UV-activatable derivative of ClpS (the targeting component of N-end rule pathway), and UV-crosslinking in intact bacterial or eukaryotic cells. 3) Studies of the yeast and mammalian UBR-family Ub ligases, including their physiological substrates; determination of the crystal structure of UBR1; and further exploration of the discoveries that the N-end rule pathway is a sensor of heme and nitric oxide. 4) Further investigations of the recent discovery that the arginyl-transferase (R-transferase) ATE1 specifically binds to a small subset of mRNAs in mammalian cells. 5) New components and functions of the deamidation branch of the N-end rule pathway: isolation, cloning and molecular genetic dissection of mouse NTAQ1, a glutamine-specific N-terminal amidase.